Clozapine N-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. This system consists of synthetic G-protein-coupled receptors, which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N-desmethylclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long–Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuated d-amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/kg CNO significantly attenuated the d-amphetamine-induced increase in evoked dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine and N-Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effects in vivo and is converted to clozapine and N-Des emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

The pedunculopontine tegmental nucleus (PPTg) is in a key position to participate in operant reinforcement via its connections with the corticostriatal architecture and the medial reticular formation. Indeed, previous work has demonstrated that rats bearing lesions of the whole PPTg are impaired when learning to make two bar presses for amphetamine reinforcement. Anterior and posterior portions of the PPTg make different anatomical connections, including preferential projections by the anterior PPTg to substantia nigra pars compacta dopamine neurons and by the posterior PPTg to ventral tegmental area dopamine neurons. We wanted to assess the effects of anterior and posterior PPTg ibotenate lesions on rats learning simple and more complex schedules of natural reinforcement. We trained rats with lesions to the anterior PPTg (n = 11) and the posterior PPTg (n = 5) [and appropriate controls (n = 15)] to bar press for food on a variety of fixed-ratio and variable-ratio reinforcement schedules and then during extinction. We found that posterior PPTg-lesioned rats bar pressed at lower rates, were slower to learn to bar press, and often had deficits characteristic of impaired learning and/or motivation. In contrast, anterior PPTg-lesioned rats learned to bar press for reinforcement at normal rates. However, they made errors of perseveration and anticipation throughout many schedules, and pressed at a higher rate than controls during extinction, deficits best characterized as reflecting disorganized response control. Together, these data suggest that the anterior PPTg and posterior PPTg (and their related circuits) contribute differently to reinforcement learning, incentive motivation, and response control, processes that are considered to malfunction in drug addiction.

Anatomically and functionally located between basal ganglia and brainstem circuitry, the pedunculopontine tegmental nucleus (PPTg) is in a pivotal position to contribute to motor behavior. Studies in primates have reported akinesia and postural instability following destruction of the PPTg. In humans, the PPTg partially degenerates in Parkinson's disease and stimulation of this region is under investigation as a possible therapeutic. Studies in rats report no crude motor impairment following PPTg lesion, although a detailed assessment of the role of the PPTg in rat motor function has not been reported. Our studies applied motor tests generally used in rodent models of Parkinson's disease to rats bearing either excitotoxic damage to all neuronal populations within PPTg, or selective destruction of the cholinergic subpopulation created with the toxin Dtx-UII. Neither lesion type altered baseline locomotion. On the rotarod, excitotoxic lesions produced a persistent impairment on the accelerating, but not fixed speed, conditions. In the vermicelli handling task (a quantitative measure of fine motor control and effective behavioral sequencing) excitotoxic lesions produced no single impairment, but globally increased the number of normal and abnormal behaviors. In contrast, depletion of cholinergic PPTg neurons produced impairment on the accelerating rotarod but no changes in vermicelli handling. Together, these results show that while PPTg lesions produce no impairment in the execution of individual motor actions, impairments emerge when the demands of the task increase. Results are discussed in terms of PPTg acting as part of a rapid action selection system, which integrates sensory information into motor output.

HighlightsThe pedunculopontine tegmental nucleus is essential for action–outcome learning.Sensitivity to instrumental contingency degradation is blocked by PPTg inactivation.Inactivation of PPTg does not change performance of previously learnt operant tasks.This is the first demonstration of a role for brainstem in action–outcome learning.Learning functions of basal ganglia extend into the deepest parts of the circuitry.